Bacillus subtilis genetic engineering strain for high yield of 4-alpha-glycosyltransferase, and construction method and application thereof

Abstract

The invention discloses a bacillus subtilis genetic engineering strain of high-yield 4-alpha-glycosyltransferase (4GT) as well as a construction method and application of the bacillus subtilis genetic engineering strain. The engineering bacterial strain is constructed by taking bacillus subtilis DB403 as a host and introducing a recombinant plasmid pHTN13 / PspovG-PspovG-SPsacB-4GT, and the engineering bacterial strain is constructed by taking bacillus subtilis DB403 as the host. According to the invention, the powerful constitutive tandem promoter PspovG-PspovG is combined with the efficient signal peptide SPsacB, and is combined with the 4GT gene, so that the secretory expression efficiency of 4GT in bacillus subtilis is remarkably improved. According to the engineering strain, under the optimized fermentation condition, the extracellular 4GT enzyme activity of shake flask fermentation can reach 88.53 U / mL and is improved by 96.21% compared with that of an original strain. The constructed engineering strain can be used for producing the 4GT enzyme preparation with low cost and high efficiency, and has important application value in the fields of starch modification, functional food development and the like.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and microbial fermentation technology, and relates to a Bacillus subtilis genetically engineered strain that produces high levels of 4-α-glycosyltransferase, its construction method and application, specifically relating to a method for constructing a Bacillus subtilis genetically engineered strain that achieves efficient secretion and expression of 4-α-glycosyltransferase (4GT) through optimization of expression elements and its application. Background Technology

[0002] 4-α-glycosyltransferase (4GT, EC 2.4.1.25) catalyzes the hydrolysis and transfer of α-1,4 glycosidic bonds in starch molecules, thereby shortening the starch backbone and lengthening the side chains. This significantly improves starch's resistance to retrogradation, solubility, and digestibility, making it a promising candidate for applications in the food, cosmetics, and pharmaceutical industries. 4GT, derived from *Thermus thermophilus* HB8 (ATCC27634), has attracted considerable attention due to its excellent thermal stability.

[0003] However, the demanding culture conditions, low enzyme activity, and high fermentation costs of naturally occurring thermophilic strains severely restrict their industrialization. Although the E. coli expression system has achieved heterologous expression of 4GT, it suffers from problems such as low enzyme activity, difficulties in purification due to intracellular expression, and the risk of endotoxin contamination, limiting its application in the food and pharmaceutical fields.

[0004] Bacillus subtilis, a recognized food-grade (GRAS) microorganism, possesses excellent protein secretion capabilities, making it an ideal host for heterologous protein expression. However, currently reported recombinant expression of 4GT in Bacillus subtilis generally shows low enzyme activity, and there is a lack of systematic synergistic optimization studies on multi-level regulatory elements such as promoters and signal peptides, resulting in the secretion and expression efficiency of 4GT failing to meet the requirements for industrial applications.

[0005] Therefore, developing a genetically engineered strain capable of efficiently secreting and expressing 4GT in Bacillus subtilis is of great significance for overcoming industrial bottlenecks and promoting the application of 4GT in related fields. Summary of the Invention

[0006] To address the technical problems of low secretion efficiency and low enzyme activity of 4-α-glycosyltransferase (4GT) expression in existing studies of Bacillus subtilis, this invention, based on existing techniques in genetic engineering and microbial fermentation, provides for the first time a high-yielding Bacillus subtilis genetically engineered strain of 4-α-glycosyltransferase, its construction method, and its application. Specifically, it provides a high-yielding Bacillus subtilis genetically engineered strain of 4-α-glycosyltransferase constructed through multi-scale, stepwise expression element optimization, its construction method, and its application in high-efficiency fermentation production. The core innovation of this invention lies in: by constructing and screening specific promoter and signal peptide libraries from the vast genetic element library of Bacillus subtilis, this invention proposes and verifies for the first time the "tandem dual promoter P..." spovG -P spovG "and "signal peptide SP sacB This unique combination exhibits a significant synergistic effect on the secretory expression of 4GT; furthermore, a precisely matched soluble starch-tryptone fermentation medium was developed. This integrated approach ultimately enabled the extracellular enzyme activity of 4GT to reach 88.53 U / mL during shake-flask fermentation, representing a 96.21% increase compared to the original optimized starting strain.

[0007] This invention provides a recombinant Bacillus subtilis engineered strain, which uses Bacillus subtilis DB403 as a host and contains a recombinant expression cassette. The recombinant expression cassette contains a promoter, a signal peptide, and genes encoding 4-α-glycosyltransferase (4GT, EC 2.4.1.25), maltotriose transferase, or 1,4-α-glucan branching enzyme. The recombinant Bacillus subtilis engineered strain can secrete and express active 4-α-glycosyltransferase, maltotriose transferase, or 1,4-α-glucan branching enzyme.

[0008] In one specific embodiment, the promoter includes P grac P 43 P 556 P spovG P yvyD P haG P lytR P sigW P xylA P yqfD P mmgA P gerBC P bsamy P phrC P gsiB P nprE P bltD One or more of the following; preferably, P spovG -P spovG .

[0009] In one specific embodiment, the signal peptide includes SP. samyQ SP sacB SP lytF SP yybN SP ybbE SP ydbK SP yddT SP phrC SP cccA SP yxiT SP yvbX P spovG One or more of the following; preferably, SP. sacB .

[0010] In one specific embodiment, the recombinant Bacillus subtilis engineered strain uses Bacillus subtilis DB403 as a host and contains a recombinant expression cassette, the recombinant expression cassette containing tandem P spovG Promoter, signal peptide SP sacB The strain is capable of secreting and expressing active 4-α-glycosyltransferase, including its coding sequence and the gene encoding 4-α-glycosyltransferase.

[0011] The recombinant expression cassette is located in one or more of the plasmids pHTN13, pHT01, pHT43, pWB980, pBE, etc.

[0012] In one specific embodiment, the recombinant expression cassette is located in plasmid pHTN13, constituting recombinant plasmid pHTN13 / P. spovG -P spovG -SP sacB -4GT.

[0013] In one specific embodiment, the present invention provides a recombinant Bacillus subtilis engineered strain that produces high levels of 4-α-glycosyltransferase. This strain uses Bacillus subtilis DB403 as a host, and the recombinant plasmid pHTN13 / P, innovatively constructed in this invention, is introduced into and expressed in the host. spovG -P spovG -SP sacB -4GT was obtained; wherein the recombinant plasmid pHTN13 / P spovG -P spovG -SP sacB -4GT contains the optimal tandem dual promoter P proposed in this invention. spovG -P spovG Optimal signal peptide SP sacB The coding sequence and the coding gene for 4-α-glycosyltransferase (4GT) derived from Thermus thermophilus HB8. The optimal signal peptide is derived from SP.samyQ SP sacB SP lytF SP yybN SP ybbE SP ydbK SP yddT SP phrC SP cccA SP yxiT SP yvbX The optimal promoter was obtained from P through screening; grac P 43 P 556 P spovG P yvyD P haG P lytR P sigW P xylA P yqfD P mmgA P gerBC P bsamy P phrC P gsiB P nprE P bltD The 4-α-glycosyltransferase was obtained through screening. The nucleotide sequence of the gene encoding the 4-α-glycosyltransferase is shown in SEQ ID NO: 1.

[0014] The present invention also provides a method for constructing the recombinant Bacillus subtilis engineered strain as described above. The method includes: using Bacillus subtilis DB403 as a host, and constructing the recombinant Bacillus subtilis engineered strain by introducing and expressing the recombinant expression cassette as described above into the host.

[0015] In one specific embodiment, the present invention also provides a method for constructing the recombinant Bacillus subtilis engineered strain as described above, the method comprising: using Bacillus subtilis DB403 as a host, and introducing and expressing the recombinant plasmid pHTN13 / P into the host. spovG -P spovG -SP sacB -4GT was used to construct the recombinant Bacillus subtilis engineered strain.

[0016] In one specific embodiment, the present invention provides a method for constructing the recombinant Bacillus subtilis engineered strain, which is achieved through the following step-by-step optimization strategy:

[0017] (1) Constructing the original expression unit:

[0018] Constructing a system containing an inductive promoter P grac Signal peptide SP samyQ The original plasmid pHTN13 / P of the 4GT genegrac -SP samyQ -4GT was transformed into Bacillus subtilis DB403 to obtain the original engineered strain.

[0019] (2) Screening and optimization of signal peptide libraries:

[0020] Using the pHTN13 / P constructed in step (1) grac -SP samyQ -4GT plasmid as the backbone, containing SP samyQ The signal peptide is replaced with various different signal peptides from the Sec or Tat pathways, and the coding sequences of the signal peptides are selected from SP. sacB SP lytF SP yybN SP ybbE SP ydbK SP yddT SP phrC SP cccA SP yxiT or SP yvbX Through fermentation verification, the optimal signal peptide SP was screened from the above signal peptides. sacB And obtained the optimized plasmid pHTN13 / P grac -SP sacB -4GT.

[0021] (3) Screening and optimization of the startup sub-library:

[0022] a) Single promoter screening: using the pHTN13 / P constructed in step (1) grac -SP samyQ Using the -4GT plasmid as a backbone, the inducible promoter P... grac They were replaced with various different compositional strong promoters, the promoters being selected from P 43 P 556 P spovG P yvyD P haG P lytR P sigW P xylA P yqfD P mmgA P gerBC P bsamy P phrC P gsiB P nprE P bltD Through fermentation verification, the optimal single promoter P was selected from the above promoters. spovG .

[0023] b) Tandem promoter enhancement: Based on step a), the optimal single promoter P obtained through screening is further enhanced. spovG Constructed as a series dual promoter P spovG -P spovG and replace P in the original plasmid grac This was used to verify its enhancement effect compared to a single promoter.

[0024] (4) Integration of optimal components:

[0025] The optimal signal peptide SP obtained in step (2) sacB With the optimal tandem promoter P obtained in step (3) spovG -P spovG Combine and replace the original plasmid pHTN13 / P in step (1). grac -SP samyQ P in -4GT grac and SP samyQ Finally, the recombinant plasmid pHTN13 / P, which integrates the optimal expression elements, was constructed. spovG -P spovG -SP sacB -4GT was transformed into Bacillus subtilis DB403 to obtain a recombinant Bacillus subtilis engineered strain that produces 4-α-glycosyltransferase as described above.

[0026] The present invention also provides a method for producing 4-α-glycosyltransferase, the method comprising: fermenting a recombinant Bacillus subtilis engineered strain as described above, and obtaining 4-α-glycosyltransferase from the fermentation broth.

[0027] In one specific embodiment, the fermentation medium used for fermentation includes a carbon source and a nitrogen source, etc.; wherein, the carbon source includes soluble starch, etc., and the nitrogen source includes tryptone, etc.

[0028] In one specific embodiment, the inoculum amount for fermentation is 1.0%-3.0% (v / v); preferably, it is 2.0% (v / v).

[0029] In one specific embodiment, the fermentation medium comprises: 1.0%-10.0% (w / v) soluble starch, 0.5%-3.0% (w / v) tryptone, 20-28 g / L yeast extract, 14-19 g / L K₂HPO₄, 1.8-2.6 g / L KH₂PO₄, etc., with water as the solvent, and a pH of 6.8-7.2; preferably, the fermentation medium comprises: 3.0% (w / v) soluble starch, 2.0% (w / v) tryptone, 24 g / L yeast extract, 16.4 g / L K₂HPO₄, 2.2 g / L KH₂PO₄, etc., with water as the solvent, and a pH of 7.0.

[0030] In one specific embodiment, the method includes a method for producing 4-α-glycosyltransferase by fermentation using the recombinant Bacillus subtilis engineered strain as described above. Specifically, the method includes: using a systematically optimized fermentation process to conduct shake-flask fermentation culture of the recombinant Bacillus subtilis engineered strain as described above, in order to ferment and produce 4-α-glycosyltransferase; wherein, the systematic optimization of the fermentation process includes, based on the initial TB medium, sequentially optimizing the inoculum age, inoculum size, glycerol concentration, carbon source type and concentration, and nitrogen source type and concentration, and determining the final optimal combination through orthogonal experiments; wherein, the initial TB medium contains: 12 g / L tryptone, 24 g / L yeast extract, 4 g / L glycerol, 16.4 g / L K2HPO4, 2.2 g / L KH2PO4, etc., with solvents including water, and a pH of 7.0; the optimal fermentation medium composition proposed in this invention includes: 3.0% (w / v) soluble starch, 2.0% (w / v) tryptone, 24 g / L yeast extract, 16.4 g / L K2HPO4, 2.2 g / L KH2PO4, etc. g / L, solvents include water, pH 7.0.

[0031] In one specific embodiment, the method includes the following steps:

[0032] a) Seed culture preparation: The recombinant Bacillus subtilis engineered strain was inoculated into LB seed medium and activated to the optimal seed age to obtain seed culture;

[0033] b) Fermentation culture: The seed culture obtained in step (a) is transferred to the optimal fermentation medium at the optimal inoculum size and cultured with shaking for 20-28 hours to obtain a fermentation broth containing high-activity 4-α-glycosyltransferase;

[0034] In step (a), the optimal seed age is 6-10 hours; more preferably, the optimal seed age is 8 hours.

[0035] In step (a), the activation culture temperature is 35-37℃; preferably, it is 37℃.

[0036] In step (b), the optimal inoculation amount is 1.0%-3.0% (v / v); more preferably, the optimal inoculation amount is 2.0% (v / v).

[0037] In step (b), the conditions for the oscillation culture include: 35-38℃, 180-220 rpm, etc.; preferably, the conditions for the oscillation culture include: 37℃, 200 rpm, etc.

[0038] In step (b), the optimal fermentation medium is determined through the following stepwise optimization process:

[0039] (1) Optimization of glycerol concentration: Based on the initial TB medium, the glycerol concentration is optimized to 2-10 g / L; preferably, it is optimized to 6-10 g / L; more preferably, the optimal glycerol concentration is 6 g / L.

[0040] (2) Carbon source replacement and optimization: Based on the optimization in step 1, the carbon source in the culture medium was replaced with soluble starch instead of glycerol, and its concentration was optimized;

[0041] The concentration of soluble starch is preferably 1.0%-10.0% (w / v); more preferably, the concentration is 2.0%-4.0% (w / v); and most preferably, the concentration is 3.0% (w / v).

[0042] (3) Nitrogen source optimization: The nitrogen source (tryptone) in the culture medium was replaced with tryptone, and its concentration was optimized;

[0043] The concentration of tryptone is preferably 0.5%-3.0% (w / v); more preferably, the concentration is 1.5%-2.5% (w / v); and most preferably, the concentration is 2.0% (w / v).

[0044] (4) Determining the optimal combination through orthogonal experiments: via L9(3 3 An orthogonal experiment was conducted to comprehensively verify the above key factors, and the final composition of the optimal fermentation medium was determined to be: soluble starch 3.0% (w / v), tryptone 2.0% (w / v), yeast extract 24 g / L, K2HPO4 16.4 g / L, KH2PO4 2.2 g / L, etc., with water as the solvent, and pH 7.0; at the same time, the optimal inoculum size for the fermentation culture was determined to be 2.0% (v / v).

[0045] The present invention also provides a fermentation medium comprising: 1.0%-10.0% (w / v) soluble starch, 0.5%-3.0% (w / v) tryptone, 20-28 g / L yeast extract, 14-19 g / L K₂HPO₄, 1.8-2.6 g / L KH₂PO₄, etc., with water as the solvent, and a pH of 6.8-7.2. Preferably, the fermentation medium comprises: 3.0% (w / v) soluble starch, 2.0% (w / v) tryptone, 24 g / L yeast extract, 16.4 g / L K₂HPO₄, 2.2 g / L KH₂PO₄, etc., with water as the solvent, and a pH of 7.0.

[0046] The present invention also provides a primer, the nucleotide sequence of which includes one or more of the nucleotide sequences shown in SEQ ID NO: 4-65.

[0047] The present invention also provides a primer pair, wherein the nucleotide sequence of the primer pair includes one or more of the nucleotide sequences shown in SEQ ID NO: 4-65.

[0048] The present invention also provides a nucleotide of a 4-α-glycosyltransferase, the nucleotide sequence of which is shown in SEQ ID NO:1. The present invention also provides a protein of a 4-α-glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO:2.

[0049] The present invention also provides a plasmid comprising pHTN13 / P grac -SP samyQ -4GT, pHTN13 / P grac -SP sacB -4GT, pHTN13 / P grac -SP lytF -4GT, pHTN13 / P grac -SP yybN -4GT, pHTN13 / P grac -SP ybbE -4GT, pHTN13 / P grac -SP ydbK -4GT, pHTN13 / P grac -SP yddT -4GT, pHTN13 / P grac -SP phrC -4GT, pHTN13 / P grac -SP cccA -4GT, pHTN13 / P grac -SP yxiT-4GT、pHTN13 / P grac -SP yvbX -4GT、pHTN13 / P 43 -SP samyQ -4GT、pHTN13 / P 556 -SP samyQ -4GT、pHTN13 / P spovG -SP samyQ -4GT、pHTN13 / P yvyD -SP samyQ -4GT、pHTN13 / P haG -SP samyQ -4GT、pHTN13 / P lytR -SP samyQ -4GT、pHTN13 / P sigW -SP samyQ -4GT、pHTN13 / P xylA -SP samyQ -4GT、pHTN13 / P yqfD -SP samyQ -4GT、pHTN13 / P mmgA -SP samyQ -4GT、pHTN13 / P gerBC -SP samyQ -4GT、pHTN13 / P bsamy -SP samyQ -4GT、pHTN13 / P phrC -SP samyQ -4GT、pHTN13 / P gsiB -SP samyQ -4GT、pHTN13 / P nprE -SP samyQ -4GT、pHTN13 / P bltD -SP samyQ -4GT、pHTN13 / P spovG -P haG -SP samyQ -4GT、pHTN13 / P spovG -P yvyD -SP samyQ -4GT、pHTN13 / P spovG -P 43 -SP samyQ -4GT、pHTN13 / P spovG -P spovG -SP samyQ -4GT、pHTN13 / P spovG -P spovG -SP sacBOne or more of -4GT, etc. The basic framework of the pHTN13 series plasmids is based on pHTN13 / P grac -SP samyQ (SEQ ID NO:3) is used as an example for demonstration. The remaining plasmids are constructed according to the methods in the embodiments below.

[0050] This invention also provides applications of the recombinant Bacillus subtilis engineered strains, methods, primers, primer pairs, and plasmids described above in the fermentation production of 4-α-glycosyltransferase, preparation of high-yield 4-α-glycosyltransferase (4GT) Bacillus subtilis genetically engineered strains, high-efficiency fermentation production, large-scale / economical production of 4GT enzyme preparations, efficient secretory expression systems, starch modification and functional food development, and in the fields of food and medicine.

[0051] In one specific embodiment of the present invention, the gene encoding the 4-α-glycosyltransferase is derived from Thermus thermophilus HB8, ATCC27634.

[0052] In one specific embodiment of the present invention, the nucleotide sequence of the gene encoding the 4-α-glycosyltransferase is shown in SEQ ID NO: 1;

[0053] The SP samyQ The signal peptide has an amino acid sequence identical to positions 1 to 31 of the α-amylase precursor protein shown in NCBI accession number AAA22192.1; the SP sacB The signal peptide has an amino acid sequence identical to positions 1 to 29 of the SacB protein shown in NCBI accession number CCD10844.1; the SP yybN The signal peptide has an amino acid sequence identical to positions 1 to 30 of the DUF2712 domain protein shown in NCBI accession number WP_009968442.1; the SP cccA The signal peptide has an amino acid sequence identical to positions 1 to 27 of the cytochrome c-550 protein shown in NCBI accession number MCY9210509.1; the SP lytF The signal peptide has an amino acid sequence identical to that shown in NCBI accession number AFG28268.1; the SP ybbE The signal peptide has an amino acid sequence identical to that shown in NCBI accession number AFG28210.1; the SP ydbK The signal peptide has an amino acid sequence identical to that shown in NCBI accession number AFG28261.1; the SP yddTThe signal peptide has an amino acid sequence identical to that shown in NCBI accession number AFG28204.1; the SP phrC The signal peptide has an amino acid sequence identical to that shown in NCBI accession number AFG28211.1; the SP yxiT The signal peptide has an amino acid sequence identical to that shown in NCBI accession number AFG28238.1; the SP yvbX The signal peptide has the same amino acid sequence as that shown in NCBI accession number AFG28232.1.

[0054] The P grac The promoter has a nucleotide sequence identical to that of positions 613110 to 613244 in the sequence shown in NCBI accession number CP133412.1; the P 43 The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP035167.1, from position 2449187 to 2449486; the P 556 The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP133412.1, from positions 3343655 to 3343709; the P spovG The promoter has a nucleotide sequence identical to positions 55546 to 55845 of the sequence shown in NCBI accession number CP133412.1; the P yvyD The promoter has a nucleotide sequence identical to positions 3926909 to 3927208 of the sequence shown in NCBI accession number CP154918.1; the P haG The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP133412.1, from position 3433573 to 3433872; the P lytR The promoter has a nucleotide sequence identical to positions 2507534 to 2507833 of the sequence shown in NCBI accession number CP116829.1; the P sigW The promoter has a nucleotide sequence identical to that of positions 188854 to 189153 in the sequence shown in NCBI accession number CP035411.1; the P xylA The promoter has a nucleotide sequence identical to positions 1734205 to 1734714 of the sequence shown in NCBI accession number CP133412.1; the P yqfDThe promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP035411.1, from positions 2481808 to 2482107; the P mmgA The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP133412.1, from position 2356834 to 2357133; the P gerBC The promoter has a nucleotide sequence identical to positions 3486142 to 3486441 of the sequence shown in NCBI accession number CP133412.1; the P bsamy The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP054050.1, from position 3852127 to 3852326; the P phrC The promoter has a nucleotide sequence identical to positions 413520 to 413819 of the sequence shown in NCBI accession number CP133412.1; the P gsiB The promoter has a nucleotide sequence identical to that of positions 478116 to 478421 in the sequence shown in NCBI accession number CP133412.1; the P nprE The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP035411.1 from position 1518759 to 1519160; the P bltD The promoter has the same nucleotide sequence as positions 2514262 to 2514562 in the sequence shown in NCBI accession number CP133412.1.

[0055] In one specific embodiment of the present invention, the nucleotide sequence described above further includes a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence; or a nucleic acid sequence having the same function as the nucleotide sequence formed by substitution, deletion or addition of one or more nucleic acid bases; or a nucleotide sequence that hybridizes with the nucleotide sequence or its full-length complement under stringent conditions; or a nucleotide sequence that is distinct from the nucleotide sequence due to the degeneracy of the genetic codon.

[0056] Compared with existing technologies, the present invention has the following advantages: It creates a highly efficient secretory expression system: The present invention systematically screens and identifies the optimal promoter P for efficient secretory expression of 4GT in Bacillus subtilis. spovG and the optimal signal peptide SP sacBFurthermore, the transcriptional level was further enhanced through a tandem promoter strategy, constructing a novel and highly efficient secretory expression system. Enzyme activity was exponentially increased: Through optimized combination of the above-mentioned elements and matching with the fermentation process, the engineered strain constructed in this invention achieved an extracellular 4GT enzyme activity of up to 88.53 U / mL in shake-flask fermentation, a 96.21% increase compared to the original strain (45.12 U / mL), solving the bottleneck problem of low enzyme activity in existing recombinant expression systems. Comprehensive protection and robust technical barriers: This invention not only protects the final optimal combination but also constructs a comprehensive patent protection network by disclosing the preferred element library (10 signal peptides and 16 promoters) in each step, effectively preventing design circumvention. Huge industrialization potential: The use of non-inducible strong promoters and low-cost fermentation raw materials, along with a simple and controllable process, lays a solid foundation for the large-scale and economical production of 4GT enzyme preparations. Attached Figure Description

[0057] Figure 1 The construction process of pHTN13-4GT was validated. Figure A shows the PCR product of 4GT; Figure B shows the double digestion product of pHTN13 with Xba I and Sma I; Figure C shows the double digestion product of recombinant plasmid pHTN13-4GT with Xba I and Sma I.

[0058] Figure 2 To validate the expression of recombinant 4GT in Bacillus subtilis DB403. Figure A is a schematic diagram of the recombinant vector pHTN13-4GT; Figure B is an SDS-PAGE of the extracellular supernatant of pHTN13-4GT / DB403 and pHTN13 / DB403 after 24 h of fermentation; Figure C is a diagram of pHTN13-4GT (6 his) / DB403 schematic diagram; Figure D is pHTN13-4GT‍(6 Western blotting images of his / DB403 and pHTN13 / DB403, with pHTN13 / DB403 as the negative control. M: Stained protein marker, with the arrow indicating the 4GT position.

[0059] Figure 3 The expression of 4GT recombinant bacteria from 0 h to 84 h was analyzed. Figure A shows the extracellular enzyme activity and OD600 analysis of the recombinant bacteria from 0 h to 84 h; Figure B shows the SDS-PAGE images of the recombinant bacteria at different locations from 0 h to 84 h, M: Stained Protein Marker, with arrows indicating the location of 4GT.

[0060] Figure 4The effects of different signal peptides on 4GT secretion and expression are shown in Figure A. Figure A is a schematic diagram of signal peptide substitution; Figure B shows the extracellular enzyme activity and OD600 analysis of recombinant bacteria fermented for 24 h with different signal peptides; Figure C shows the extracellular protein expression of different recombinant bacteria fermented for 24 h by SDS-PAGE analysis. M: Stained Protein Marker, the dashed box indicates the optimal selection, and the arrow indicates the position of 4GT.

[0061] Figure 5 The effect of a single promoter on 4GT expression level is shown in Figure A. Figure A is a schematic diagram of different promoter substitutions; Figure B shows the 24-h extracellular enzyme activity and OD600 analysis mediated by different promoters; Figure C shows the SDS-PAGE analysis of extracellular expression in different recombinant bacteria at 24 h. M: Stained Protein Marker, the dashed box indicates the optimal choice, and the arrow indicates the position of 4GT.

[0062] Figure 6 The effect of tandem promoters on 4GT expression levels is shown in Figure A. Figure A is a schematic diagram of tandem promoter substitution; Figure B shows the 24-h extracellular enzyme activity and OD600 analysis mediated by different tandem promoter combinations; Figure C shows the SDS-PAGE analysis of extracellular expression in different recombinant bacteria at 24 h. M: Stained Protein Marker, the dashed box indicates the optimal choice, and the arrow indicates the position of 4GT.

[0063] Figure 7 The effect of the optimal combination of expression elements on 4GT expression level is shown in Figure A. Figure A is a schematic diagram of the substitution of the optimal combination of expression elements; Figure B shows the 24-h extracellular enzyme activity and OD600 analysis mediated by different element combinations; Figure C shows the SDS-PAGE analysis of extracellular expression in different recombinant bacteria at 24 h, M: Stained Protein Marker, with arrows indicating the position of 4GT.

[0064] Figure 8 The effect of seed age on 4GT expression level. Figure A shows the effect of different seed ages on 4GT extracellular enzyme activity and OD600 at 24 h; Figure B shows the extracellular expression level at 24 h as analyzed by SDS-PAGE, M: Stained Protein Marker, with arrows indicating the location of 4GT.

[0065] Figure 9 The effect of inoculum size on 4GT expression level is shown in Figure A. Figure A shows the effect of different inoculum sizes on 4GT extracellular enzyme activity and OD600 at 24 h; Figure B shows the extracellular expression level at 24 h as analyzed by SDS-PAGE. M: Stained Protein Marker, with arrows indicating the location of 4GT.

[0066] Figure 10The effect of glycerol concentration on 4GT expression level is shown in Figure A. Figure A shows the effect of different glycerol concentrations on 4GT extracellular enzyme activity and OD600 at 24 h; Figure B shows the extracellular expression level at 24 h as analyzed by SDS-PAGE. M: Stained Protein Marker, with arrows indicating the location of 4GT.

[0067] Figure 11 The effects of different carbon sources and their amounts on 4GT enzyme activity were shown in Figure A. Figure A shows the effect of different carbon sources on 4GT extracellular enzyme activity and OD600; Figure B shows the extracellular expression level at 24 h as analyzed by SDS-PAGE; Figure C shows the effect of different concentrations of soluble starch on 4GT extracellular enzyme activity and OD600 at 24 h; Figure D shows the extracellular expression level at 24 h as analyzed by SDS-PAGE. M: Stained Protein Marker, with arrows indicating the location of 4GT.

[0068] Figure 12 The effects of different nitrogen sources and their amounts on 4GT enzyme activity were shown in Figure A. Figure A shows the effects of different nitrogen sources on 4GT extracellular enzyme activity and OD600; Figure B shows the extracellular expression level at 24 h as analyzed by SDS-PAGE; Figure C shows the effects of different concentrations of tryptone on 4GT extracellular enzyme activity and OD600 at 24 h; Figure D shows the extracellular expression level at 24 h as analyzed by SDS-PAGE. M: Stained Protein Marker, with arrows indicating the location of 4GT.

[0069] Figure 13 For L9 (3 3 Table of orthogonal experimental results.

[0070] Figure 14 This is for the analysis of orthogonal experimental data. Figure A shows the inter-subject effects detection table; Figure B shows the factor level analysis.

[0071] Figure 15 The fermentation of the 4GT recombinant strain before and after orthogonal optimization was verified. Figure A shows the extracellular enzyme activity and OD600 of the recombinant strain before and after optimization at 24 h; Figure B shows the extracellular expression level at 24 h analyzed by SDS-PAGE, M: Stained Protein Marker, with the arrow indicating the location of 4GT. Detailed Implementation

[0072] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. The processes, conditions, experimental methods, etc., for implementing the present invention, except for those specifically mentioned below, are all common knowledge and general knowledge in the field, and the present invention does not have any particular limitations.

[0073] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0074] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0075] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0076] This invention discloses a genetically engineered Bacillus subtilis strain that produces high levels of 4-α-glycosyltransferase (4GT), its construction method, and its applications. This engineered strain uses Bacillus subtilis DB403 as the host and incorporates the recombinant plasmid pHTN13 / P. spovG -P spovG -SP sacB It is constructed using -4GT. This invention utilizes a powerful constitutive tandem promoter P spovG -P spovG With high-efficiency signal peptide SP sacB By combining the strains with the 4GT gene, the secretory expression efficiency of 4GT in Bacillus subtilis was significantly improved. Under optimized fermentation conditions, the extracellular 4GT enzyme activity of this engineered strain reached 88.53 U / mL during shake-flask fermentation, an increase of 96.21% compared to the original strain. The engineered strain constructed in this invention can be used for low-cost and high-efficiency production of 4GT enzyme preparations, and has important application value in the fields of starch modification and functional food development.

[0077] All primers used in the embodiments of this invention were synthesized by Qingke Biotechnology Co., Ltd., and their sequence information is summarized in Table 1 below. The primers mentioned in subsequent embodiments correspond to Table 1.

[0078] Table 1: List of primer sequences used in this invention

[0079]

[0080]

[0081]

[0082] The strains and plasmids used in the embodiments of this invention are shown in Table 2 below:

[0083] Table 2

[0084]

[0085] The basic backbone of the pHTN13 series plasmids constructed in this invention is pHTN13 / P. grac -SP samyQ As shown in SEQ ID NO:3, the remaining plasmids were constructed according to the methods described in the examples below.

[0086] The reagents used in the embodiments of this invention are as follows:

[0087] Common reagents such as sodium chloride, K2HPO4, and KH2PO4 were purchased from Sinopharm Chemical Reagent Co., Ltd.; molecular biology experimental reagents such as the San-Prep column DNA gel recovery kit, the San-Prep column PCR product purification kit, and the Hieff Clone™ Plus One Step Cloning Kit were purchased from Sangon Biotech Co., Ltd., Shanghai Yisheng Biotechnology Co., Ltd., and Nanjing Novizan Biotechnology Co., Ltd.; and the GOD-POD glucose concentration kit was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0088] Example 1: Construction and functional verification of the original expression strain

[0089] (1) Original expression plasmid pHTN13 / P grac -SP samyQ -4GT construction

[0090] In this invention, the original expression plasmid pHTN13 / P grac -SP samyQThe construction of -4GT was achieved through the following steps: First, using the pUC19-4GT plasmid containing the 4-α-glycosyltransferase (4GT) gene from *Thermophilus thermophilus* HB8 as a template, the coding region of the 4GT gene was amplified by polymerase chain reaction (PCR) using the specific primers GT-F and GT-R listed in Table 1. The PCR reaction system consisted of: 25 μL of 2×Fast Pfu Master Mix (quick Load), 1.5 μL of upstream primer GT-F, 1.5 μL of downstream primer GT-R, approximately 100 ng of template DNA, and ddH2O to a total volume of 50 μL. The PCR program was set as follows: pre-denaturation at 95℃ for 3 min; followed by 30 cycles of amplification, each cycle consisting of denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min; and finally, a final extension at 72℃ for 5 min. After completion, the sample was stored at 4℃. After verification by agarose gel electrophoresis, the PCR products were recovered using the San-Prep column-based PCR product purification kit. Simultaneously, the *E. coli*-*Bacillus subtilis* shuttle vector pHTN13 was linearized and double-digested with Xba I and Sma I restriction endonucleases. The digestion reaction mixture contained 1–2 μg of pHTN13 plasmid, 5 μL of 10×Digest Buffer, 1 μL of Xba I, and 1 μL of Sma I, with ddH2O added to a final volume of 50 μL. The mixture was incubated at 37°C for 1–2 h, and the linearized vector fragment was recovered by gel electrophoresis after the reaction. Subsequently, the purified 4GT gene fragment was ligated to the linearized pHTN13 vector via homologous recombination. The recombination reaction mixture contained approximately 100 ng of the linearized pHTN13 vector, approximately 50 ng of the 4GT PCR product, and 5 μL of 2×Hieff Clone MultiSEnzyme Premix, with ddH2O added to a final volume of 10 μL. The mixture was incubated at 50°C for 30 min. The homologous recombination product was transformed into *E. coli* DH5α competent cells. The specific steps were as follows: 10 μL of the recombination product and 100 μL of competent cells were incubated on ice for 30 min, followed by heat shock at 42℃ for 90 s, and then incubated on ice for 2 min. Subsequently, 900 μL of antibiotic-free LB broth was added, and the cells were incubated at 37℃ with shaking at 200 rpm for 1 h for recovery. Finally, an appropriate amount of bacterial culture was plated onto LB agar plates containing 100 μg / mL ampicillin and incubated upside down at 37℃ for 12–16 h. Single colonies grown on the plates were picked, and after verification by colony PCR and double enzyme digestion of the plasmid, they were sent for sequencing to confirm that the 4GT gene had been correctly inserted into the pHTN13 vector. grac Promoters and SPs samyQ Downstream of the signal peptide sequence, the correct original recombinant plasmid was obtained and named pHTN13 / P. grac-SP samyQ -4GT. The carrier construction process is as follows: Figure 1 As shown.

[0091] (2) Obtaining the original recombinant strain and verifying its enzyme activity

[0092] The recombinant plasmid pHTN13 / P constructed and verified as correct was used. grac -SP samyQ -4GT was introduced into the host bacterium Bacillus subtilis DB403 to obtain an original engineered strain capable of secreting and expressing 4GT. First, competent cells of Bacillus subtilis DB403 were prepared: DB403 strains were streaked on LB plates and cultured at 37°C for 12 h. Single colonies were picked and inoculated into 10 mL of LB liquid medium and cultured at 37°C with shaking at 200 rpm for 12 h. Then, 2% of the inoculum was transferred to 8 mL of SPI medium and cultured until the bacterial culture reached OD. 600 The pH value reached approximately 1.0; 200 μL of this bacterial culture was mixed with 2 mL of preheated SPII medium and cultured at 37°C and 150 rpm for 90 min with shaking; then 20 μL of 100 mM EGTA solution was added, and the culture was continued with shaking for 10 min. After completion, the culture was aliquoted into 400 μL tubes to obtain competent cells. For transformation, 0.8–1.2 μg of plasmid pHTN13 / P was used. grac -SP samyQ -4GT was added to one tube of competent cells, mixed well, and cultured at 37°C and 220 rpm with shaking for 60 min. 100 μL of preheated LB medium was added, mixed well, and centrifuged at 5000 rpm for 5 min. Most of the supernatant was discarded, and the bacterial pellet was resuspended in the remaining liquid. The pellet was then spread onto LB agar plates containing 25 μg / mL chloramphenicol and cultured at 37°C for 18 h. After transformants grew on the plates, single colonies were picked for plasmid extraction and PCR verification, confirming successful transformation of the recombinant plasmid into strain DB403. This strain was named pHTN13 / P. grac -SP samyQ -4GT / DB403. To verify the expression ability of this original strain, fermentation culture and enzyme activity assay were performed: A single colony that had been verified was inoculated into LB liquid medium containing 25 μg / mL chloramphenicol and activated at 37°C and 200 rpm for 6 h. Then, it was transferred at a 1% inoculation rate to TB fermentation medium containing the same concentration of chloramphenicol and cultured for 6 h until OD500 reached the target value. 600 The initial concentration was 0.6 h. IPTG was added to a final concentration of 1 mM for induction, and the mixture was cultured for another 24 h under the same conditions. After fermentation, the fermentation broth was centrifuged at 8000 rpm for 10 min, and the supernatant was collected as the crude enzyme solution. The method for determining 4GT enzyme activity is as follows:

[0093] a) Reagent preparation:

[0094] 10 mM MES buffer (pH 6.5): Accurately weigh 2.13 g of MES (2-morpholinoethanesulfonic acid monohydrate) and dissolve it in approximately 900 mL of ultrapure water. After stirring to dissolve, adjust the pH to 6.5 with 1 M NaOH solution, and finally bring the volume to 1 L.

[0095] Enzyme activity assay substrate solution: Accurately weigh 1.0 g of maltotriose, dissolve it in the above 10 mM MES buffer (pH 6.5), and bring the volume to 100 mL. Mix thoroughly.

[0096] Glucose standard solution: Use the standard provided in the commercial GOD-POD glucose assay kit, or prepare a glucose standard solution of known concentration to create a standard curve.

[0097] b) Enzymatic reaction and assay:

[0098] Add 25 μL of appropriately diluted fermentation supernatant to 100 μL of enzyme activity assay substrate solution, mix well, and react precisely in a 60°C water bath for 40 min. Immediately afterwards, heat in a 100°C metal bath for 5 min to terminate the reaction. After cooling the reaction solution under running water, take an appropriate amount of the reaction solution and determine the glucose content according to the instructions of the commercial GOD-POD glucose kit. A substrate solution without enzyme solution is also provided as a blank control.

[0099] c) Enzyme activity calculation:

[0100] One unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of glucose per minute under the above-described assay conditions.

[0101] The results showed that the original engineered strain had 4GT activity, with an extracellular enzyme activity of 45.12 U / mL, demonstrating the effectiveness of the basic expression system and providing a starting point and control benchmark for subsequent optimization.

[0102] To confirm the successful expression of the target protein, the crude enzyme solution was analyzed by SDS-PAGE gel electrophoresis. The results showed a specific band at approximately 57.2 kDa, consistent with the theoretical molecular weight of 4GT, while the empty vector control group did not exhibit this band, preliminarily confirming the secretory expression of 4GT. Experimental results are shown below. Figure 2 B.

[0103] To further confirm this, the present invention constructed a recombinant plasmid pHTN13 / P carrying a 6×histidine tag at its carboxyl terminus. grac -SP samyQ -4GT （6*his）The recombinant strain was obtained by introducing it into the DB403 strain using the same method. The supernatant from the post-induction fermentation of this strain was collected and analyzed by SDS-PAGE and Western blotting. Detection was performed using mouse anti-His tag primary antibody and the corresponding enzyme-labeled secondary antibody. The results showed a specific immunoreaction band at the same molecular weight, confirming that this band was the target protein 4GT, demonstrating the successful secretory expression of recombinant 4GT. Experimental results are shown below. Figure 2 D.

[0104] To determine the optimal enzyme production cycle of this original strain, fermentation metabolic curves were analyzed from 0 to 84 hours. Samples were taken at specific time intervals to measure cell density and extracellular 4GT enzyme activity. The results showed that the cells reached a stable growth phase at approximately 24 hours of culture; extracellular 4GT enzyme activity increased continuously in the early stages of culture, reaching a peak of 45.12 U / mL at 24 hours, and then gradually decreased with prolonged culture time. These results clearly indicate that 24 hours is the optimal fermentation harvest time. Experimental results are shown below. Figure 3 .

[0105] Based on the above verification, the original engineered strain pHTN13 / P was confirmed. grac -SP samyQ -4GT / DB403 can effectively secrete and express catalytically active 4GT protein, and its optimal fermentation period is 24 hours. All subsequent fermentation and screening experiments on this strain were conducted under this 24-hour culture condition.

[0106] Example 2: Screening and Optimization of Signal Peptides

[0107] The original plasmid pHTN13 / P was constructed and validated as an effective plasmid according to Example 1 of the present invention. grac -SP samyQ -4GT was used as the backbone for signal peptide optimization. Plasmid construction, Bacillus subtilis transformation, and fermentation enzyme activity assay were all performed according to the methods described in Example 1 of this invention.

[0108] Specifically, the signal peptide-specific primers with homologous arms listed in Table 1 were used to pair SP. sacB -F / SP sacB -R、SP lytF -F / SP lytF -R、SP yybN -F / SP yybN -R、SP ybbE -F / SP ybbE -R、SP ydbK -F / SP ydbK -R、SP yddT -F / SP yddT -R、SP phrC -F / SP phrC-R、SP cccA -F / SP cccA -R、SP yxiT -F / SP yxiT -R、SP yvbX -F / SP yvbX -R, SP was amplified by PCR respectively. sacB SP lytF SP yybN SP ybbE SP ydbK SP yddT SP phrC SP cccA SP yxiT SP yvbX Ten signal peptides were identified. The original plasmid backbone pHTN13 / P was digested with Pst I and BamHI restriction endonucleases. grac -SP samyQ -4GT, and then precisely replaced the original plasmid backbone pHTN13 / P with different signal peptide coding sequence fragments obtained by homologous recombination reaction. grac -SP samyQ SP in -4GT samyQ Signal peptide sequences were obtained, leading to the successful construction of a series of signal peptide-optimized plasmids pHTN13 / P. grac -SP sacB -4GT, pHTN13 / P grac -SP lytF -4GT, pHTN13 / P grac -SP yybN -4GT, pHTN13 / P grac -SP ybbE -4GT, pHTN13 / P grac -SP ydbK -4GT, pHTN13 / P grac -SP yddT -4GT, pHTN13 / P grac -SP phrC -4GT, pHTN13 / P grac -SP cccA -4GT, pHTN13 / P grac -SP yxiT -4GT, pHTN13 / P grac -SP yvbX -4GT. The optimized plasmids of each signal peptide constructed above were transformed into Bacillus subtilis DB403 to obtain the corresponding recombinant engineered strains. Each strain was subjected to shake-flask fermentation, and the extracellular 4GT enzyme activity was measured after 24 h of fermentation. Through comparative analysis, SP was selected as the optimal signal peptide with the highest enzyme activity from among the 10 signal peptides.sacB Equipped with the optimal signal peptide SP sacB engineered strain pHTN13 / P grac -SP sacB -4GT / DB403 exhibited an extracellular enzyme activity of 62.43 U / mL, representing a 38.36% increase compared to the original strain (45.12 U / mL). Experimental results are shown below. Figure 4 .

[0109] Example 3: Promoter Screening and Optimization

[0110] The original plasmid pHTN13 / P was constructed and validated as described in Example 1 of this invention. grac -SP samyQ -4GT was used as the backbone for promoter optimization. Plasmid construction, Bacillus subtilis transformation, and fermentation enzyme activity assays were all performed according to the methods described in Example 1 of this invention. However, a key improvement exists: since the promoters screened in this example are all constitutive promoters, their driving gene transcription does not require an external inducing agent. Therefore, IPTG induction is unnecessary during fermentation. This improvement significantly simplifies the fermentation process and reduces production costs.

[0111] (1) Single promoter screening:

[0112] Using the promoter-specific primers with homologous arms listed in Table 1, P was amplified by PCR. 43 P 556 P spovG P yvyD P haG P lytR P sigW P xylA P yqfD P mmgA P gerBC P bsamy P phrC P gsiB P nprE P bltD Sixteen constitutive strong promoter sequences (their nucleotide sequences are shown in SEQ ID NO: 14-29). The original plasmid backbone pHTN13 / P was digested with Not I and Spe I restriction endonucleases. grac -SP samyQ -4GT, and then the amplified different promoter fragments were used to replace the inducible promoter P in the original plasmid through homologous recombination. grac Thus, a series of single-promoter optimized plasmids pHTN13 / P were successfully constructed. 43 -SP samyQ -4GT, pHTN13 / P 556-SP samyQ -4GT, pHTN13 / P spovG -SP samyQ -4GT, pHTN13 / P yvyD -SP samyQ -4GT, pHTN13 / P haG -SP samyQ -4GT, pHTN13 / P lytR -SP samyQ -4GT, pHTN13 / P sigW -SP samyQ -4GT, pHTN13 / P xylA -SP samyQ -4GT, pHTN13 / P yqfD -SP samyQ -4GT, pHTN13 / P mmgA -SP samyQ -4GT, pHTN13 / P gerBC -SP samyQ -4GT, pHTN13 / P bsamy -SP samyQ -4GT, pHTN13 / P phrC -SP samyQ -4GT, pHTN13 / P gsiB -SP samyQ -4GT, pHTN13 / P nprE -SP samyQ -4GT, pHTN13 / P bltD -SP samyQ -4GT.

[0113] The optimized plasmids of each single promoter were transformed into Bacillus subtilis DB403 to obtain the corresponding recombinant engineered strains. Each strain was subjected to shake-flask fermentation, and its extracellular 4GT enzyme activity was measured. Through comparative analysis, the optimal single promoter was selected from 16 promoters as P. spovG Equipped with this optimal single-starter P spovG engineered strain pHTN13 / P spovG -SP samyQ -4GT / DB403 exhibited an extracellular enzyme activity of 60.56 U / mL, representing a 34.22% increase compared to the original strain (225 U / mL). Experimental results are shown below. Figure 5 .

[0114] (2) Tandem promoter enhancement:

[0115] To obtain the optimal single promoter P spovG Based on this, a tandem dual promoter P was constructed using overlap extension PCR technology.spovG -P haG P spovG -P yvyD P spovG -P 43 P spovG -P spovG Fragment. Using the same enzyme digestion (Not I / SpeI) and homologous recombination method as in step (1), the tandem promoter fragment was again digested with the original plasmid pHTN13 / P. grac -SP samyQ -4GT is the skeleton, replacing the P within it. grac The promoter was used to construct the enhanced transcription plasmid pHTN13 / P. spovG -P haG -SP samyQ -4GT, pHTN13 / P spovG -P yvyD -SP samyQ -4GT, pHTN13 / P spovG -P 43 -SP samyQ -4GT, pHTN13 / P spovG -P spovG -SP samyQ -4GT.

[0116] Fermentation validation showed that the engineered strain pHTN13 / P, equipped with a tandem dual promoter, was effective. spovG -P spovG -SP samyQ The extracellular enzyme activity of -4GT / DB403 reached 62.96 U / mL, compared to single-promoter P spovG (60.56 U / mL), transcription and expression levels were further enhanced. See the experimental results below. Figure 6 .

[0117] Example 4: Integration of Optimal Components and Acquisition of High-Yielding Strains

[0118] The optimal results obtained in Examples 1-3 of the present invention (preceding steps) were integrated. Plasmid construction, Bacillus subtilis transformation, and fermentation enzyme activity determination were all performed according to the method described in Example 1 of the present invention. The plasmid pHTN13 / P containing a strong tandem promoter constructed in Example 3 of the present invention was used. spovG -P spovG -SP samyQ -4GT was used as the backbone, and Pst I and BamHI were used for enzyme digestion. Simultaneously, the optimal signal peptide SP obtained from Example 2 of this invention was screened. sacB The coding sequence was amplified by PCR and contained homologous arms. The SP was then converted to a homologous recombination reaction. sacB Replace SP on the backbone plasmidsamyQ Finally, the recombinant plasmid pHTN13 / P, which integrates the optimal expression elements, was obtained. spovG -P spovG -SP sacB -4GT was obtained and verified by sequencing. This highest quality grain was then transformed into Bacillus subtilis DB403 to obtain the high-yielding engineered strain pHTN13 / P. spovG -P spovG -SP sacB -4GT / DB403.

[0119] To verify the effect of optimal component integration, the extracellular enzyme activity of the strain was measured after shake-flask fermentation. The results showed that the extracellular 4GT enzyme activity of the integrated strain reached 70.22 U / mL, an increase of 55.63% compared to the original system. This enzyme activity level was not only significantly higher than the original strain (45.12 U / mL) in Example 1 of this invention, but also higher than the intermediate strains that only optimized the signal peptide or only optimized the promoter, demonstrating that combining the optimal tandem promoter with the optimal signal peptide produced a synergistic effect, which is key to achieving efficient secretory expression of 4GT. Experimental results are shown below. Figure 7 .

[0120] Example 5: System optimization and high-yield verification of fermentation process

[0121] The high-yield engineered strain pHTN13 / P obtained in Example 4 of this invention spovG -P spovG -SP sacB Using -4GT / DB403 as the research object, the fermentation process was systematically optimized based on the initial TB medium (composition: tryptone 12 g / L, yeast extract 24 g / L, glycerol 4 g / L, K2HPO4 16.4 g / L, KH2PO4 2.2 g / L, solvent including water, pH adjusted to 7.0) to further improve the extracellular yield of 4GT. The initial fermentation conditions were as follows: the recombinant bacteria were streaked in four zones on LB agar plates containing 25 μg / mL chloramphenicol and incubated upside down at 37℃ for 14 hours. Then, morphologically typical single colonies were inoculated into 10 mL of LB liquid medium containing 25 μg / mL chloramphenicol and cultured on a shaker (200 rpm) at 37℃ for 6 hours for primary scale-up. Next, the bacterial culture was transferred at a 1% inoculum to 30 mL of TB medium containing the same concentration of chloramphenicol for further scale-up, maintaining the same temperature and shaking conditions.

[0122] (1) Single-factor optimization experiment

[0123] a) Seed age optimization:

[0124] The activated bacterial strain was cultured for 4 h, 6 h, 8 h, 10 h, 12 h, and 14 h, respectively, and then transferred to the initial TB medium at an inoculum rate of 1% (v / v). After incubation at 37℃ and 200 rpm for 24 h, enzyme activity was measured. The results showed that when the inoculum age was 8 h, the cells were in the late logarithmic growth phase, exhibiting the best physiological state and yielding the highest enzyme activity in the fermentation broth. Therefore, the optimal inoculum age was determined to be 8 h. Experimental results are shown below. Figure 8 .

[0125] b) Optimization of vaccination volume:

[0126] Seed culture at the optimal inoculum age (8 h) was transferred to the initial TB medium at inoculum amounts of 0.2%, 0.5%, 1.0%, 2.0%, 2.5%, and 3.0% (v / v), respectively. Enzyme activity was measured after incubation at 37℃ and 200 rpm for 24 h. The results showed that the coupling efficiency between cell growth and product synthesis was optimal at an inoculum amount of 2.0%, resulting in peak enzyme activity. Therefore, the optimal inoculum amount was determined to be 2.0%. Experimental results are shown below. Figure 9 .

[0127] c) Glycerol concentration optimization:

[0128] Based on the initial TB medium, glycerol concentrations were adjusted to 2, 4, 6, 8, and 10 g / L, and fermented at 37℃ and 200 rpm for 24 h. The results showed no significant difference in enzyme activity at concentrations of 6 g / L, 8 g / L, and 10 g / L, and all were higher than the initial concentration (4 g / L). To save costs, the optimal glycerol concentration was determined to be 6 g / L. Experimental results are shown below. Figure 10 .

[0129] d) Carbon source optimization:

[0130] The carbon source (6 g / L glycerol) in the optimized basal medium (TB medium) of step c) was replaced by sucrose, lactose, maltose, xylose, mannose, soluble starch, and potato starch at the same addition amount, and fermented at 37℃ and 200 rpm for 24 h. The results showed that the strain had the strongest enzyme production capacity when using soluble starch as the carbon source, and the enzyme activity was significantly higher than that of other carbon sources. Further optimization of the concentration of soluble starch (gradient of 1.0%-10.0%, w / v) revealed that the enzyme activity reached its maximum value when the concentration was 3.0%. Therefore, the optimal carbon source was determined to be 3.0% soluble starch. The experimental results are shown in […]. Figure 11 .

[0131] e) Nitrogen source optimization:

[0132] The nitrogen source (tryptone) in the initial TB medium was replaced with yeast peptone FP326, yeast peptone FP103, yeast extract FM888, yeast extract FM803, tryptone, soybean peptone, fish meal peptone, and polypeptone at the same addition level (1.2%, w / v), and fermented at 37℃ and 200 rpm for 24 h. The results showed that the enzyme activity was highest and the protein band was single when tryptone was used as the nitrogen source. Further optimization of the tryptone concentration (gradient 0.5%-3.0%, w / v) revealed that the enzyme activity reached its peak at a concentration of 2.0%. Therefore, the optimal nitrogen source was determined to be 2.0% tryptone. Experimental results are shown below. Figure 12 .

[0133] (2) Determine the optimal combination through orthogonal experiments

[0134] Based on the results of the above single-factor experiments, three key factors were selected: carbon source concentration (soluble starch), nitrogen source concentration (tryptone), and inoculum size. Three levels were selected for each factor, and an L9 (3) inoculum design was implemented. 3 An orthogonal experimental design was used to optimize the enzyme activity, with extracellular enzyme activity after fermentation at 37℃ and 200 rpm for 24 h as the evaluation index. Analysis of variance showed that nitrogen source concentration had the most significant impact on enzyme activity. Through intuitive analysis and calculation, the optimal combination of factors was determined to be: soluble starch 3.0%, tryptone 2.0%, and inoculum size 2.0%. Experimental results are shown below. Figure 13 , 14 .

[0135] (3) Optimal process verification

[0136] Based on the optimal combination determined by orthogonal experiments, the final fermentation medium was prepared as follows: tryptone 20 g / L, yeast extract 24 g / L, soluble starch 30 g / L, K2HPO4 16.4 g / L, KH2PO4 2.2 g / L, with water as the solvent, and the pH was adjusted to 7.0.

[0137] Under these optimal culture medium and process conditions (8 h seed age, 2.0% inoculum), after 24 h of shake-flask fermentation at 37°C and 200 rpm, enzyme activity was measured according to the method described in Example 1 of this invention. The results showed that the extracellular 4GT enzyme activity of the engineered strain stably reached 88.53 U / mL. This value represents a 26.08% increase compared to the enzyme activity (70.22 U / mL) of the engineered strain in Example 4 after only genetic modification and 24 h of shake-flask fermentation at 37°C and 200 rpm, and a 96.21% increase compared to the enzyme activity (45.12 U / mL) of the original strain in Example 1 after 24 h of shake-flask fermentation at 37°C and 200 rpm. This fully demonstrates the significant effectiveness of the synergistic optimization of genetic modification and fermentation process. Experimental results are shown below. Figure 15.

[0138] After fermentation, the fermentation supernatant obtained by centrifugation is a high-activity 4GT crude enzyme solution, which can be used directly for subsequent applications or as a raw material for purification.

[0139] As used in this invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0140] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0141] As used in this invention, the term "about" when applied to a value indicates that a slight degree of imprecision is permissible in the calculation or measurement of the value (the accuracy of the value by some means; approximately or reasonably close to the value; almost). If, for some reason, the imprecision specified by "about" is not understood in this conventional sense in the art, then "about" as used in this invention at least indicates variation that may be caused by conventional methods of measuring or using such parameters.

[0142] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.

[0143] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0144] In this invention, the sequence information is as follows:

[0145] The coding gene sequence of SEQ ID NO: 1: 4GT

[0146]

[0147] Protein sequence of SEQ ID NO: 2: 4GT

[0148] MELPRAFGLLLHPTSLPGPYGVGVLGQEARDFLRFLLKEAGGRYWQVLPLGPTGYGDSPYQSFSAFAGNPYLIDLRPLAERGYVRLEDPGFPQGRVDYGLLYAWKWPALKEAFRGFKEKASPEERE AFAAFREREAWWLEDYALFMALKGAHGGLPWNRWPLPLRKREEKALREAKSALAEEVAFHAFTQWLFFRQWGALKAEAEALGIRIIGDMPIFVAEDSAEVWAHPEWFHLDEEGRPTVVAGVPPDY FSETGQRWGNPLYRWDVLEREGFSFWIRRLEKALELFHLVRIDHFRGFEAYWEIPASCPTAVEGRWVKAPGEKLFQKIQEVFGEVPVLAEDLGVITPEVEALRDRFGLPGMKVLQFAFDDGMENP FLPHNYPAHGRVVVYTGTHDNDTTLGWYRTATPHEKAFMARYLADWGITFREEEEVPWALMHLGMKSVARLAVYPVQDVLALGSEARMNYPGRPSGNWAWRLLPGELSPEHGARLRAMAEATERL

[0149] SEQ ID NO: 3: pHTN13 / P grac -SP samyQ Basic plasmid sequence

[0150] pHTN13 / P in this invention grac -SP samyQ The complete sequence of the basic plasmid is shown below. The addition of the 4GT gene was performed according to the method in Example 1. The replacement of different signal peptides was performed according to the method in Example 2. The replacement of different promoters was performed according to the method in Example 3. The integration of the optimal signal peptide and promoter was performed according to the method in Example 4.

[0151]

Claims

1. A recombinant Bacillus subtilis engineered strain, characterized in that, The strain uses Bacillus subtilis DB403 as a host and contains a recombinant expression cassette. The recombinant expression cassette contains a promoter, a signal peptide, and a gene encoding 4-α-glycosyltransferase, maltotriose transferase, or 1,4-α-glucan branching enzyme. The strain is capable of secreting and expressing active 4-α-glycosyltransferase, maltotriose transferase, or 1,4-α-glucan branching enzyme. in, The promoter includes P grac P 43 P 556 P spovG P yvyD P haG P lytR P sigW P xylA P yqfD P mmgA P gerBC P bsamy P phrC P gsiB P nprE P bltD One or more of the following; The signal peptide includes SP. samyQ SP sacB SP lytF SP yybN SP ybbE SP ydbK SP yddT SP phrC SP cccA SP yxiT SP yvbX P spovG One or more of them.

2. The recombinant Bacillus subtilis engineered strain according to claim 1, characterized in that, The strain uses Bacillus subtilis DB403 as a host and contains a recombinant expression cassette, which contains tandem P... spovG Promoter, signal peptide SP sacB The strain is capable of secreting and expressing an active 4-α-glycosyltransferase, including its coding sequence and the gene encoding 4-α-glycosyltransferase.

3. The recombinant Bacillus subtilis engineered strain according to claim 1, characterized in that, The recombinant expression cassette is located in one or more of the plasmids pHTN13, pHT01, pHT43, pWB980, and pBE.

4. A method, characterized in that, The method includes one or more of the following: (1) A method for constructing a recombinant Bacillus subtilis engineered strain according to any one of claims 1-3, the method comprising: using Bacillus subtilis DB403 as a host, and constructing the recombinant Bacillus subtilis engineered strain by introducing and expressing the recombinant expression cassette according to any one of claims 1-3 into the host; (2) A method for producing 4-α-glycosyltransferase, the method comprising: fermenting a recombinant Bacillus subtilis engineered strain as described in any one of claims 1-3, and obtaining 4-α-glycosyltransferase from the fermentation broth.

5. The method according to claim 4, characterized in that, In (2), the fermentation medium used for fermentation contains a carbon source and a nitrogen source; wherein the carbon source contains soluble starch and the nitrogen source contains tryptone; and / or, the inoculum amount for fermentation is 1.0%-3.0%.

6. The method according to claim 4, characterized in that, In (2), the fermentation medium comprises: 1.0%-10.0% soluble starch, 0.5%-3.0% tryptone, 20-28 g / L yeast extract, 14-19 g / L K2HPO4, 1.8-2.6 g / L KH2PO4, and water as a solvent, with a pH of 6.8-7.

2.

7. A fermentation culture medium, characterized in that, The fermentation medium comprises: 1.0%-10.0% soluble starch, 0.5%-3.0% tryptone, 20-28 g / L yeast extract, 14-19 g / L K2HPO4, 1.8-2.6 g / L KH2PO4, and solvents including water, with a pH of 6.8-7.

2.

8. A substance characterized in that, The substance includes one or more of the following: (1) A primer, the nucleotide sequence of said primer comprising one or more of the nucleotide sequences shown in SEQ ID NO: 4-65; (2) A primer pair, wherein the nucleotide sequence of the primer pair comprises one or more of the nucleotide sequences shown in SEQ ID NO: 4-65; (3) A nucleotide relating to 4-α-glycosyltransferase, the nucleotide sequence of which is shown in SEQ ID NO:1; (4) A protein relating to 4-α-glycosyltransferase, the amino acid sequence of said protein being shown in SEQ ID NO:2; (5) A plasmid comprising pHTN13 / P grac -SP samyQ -4GT, pHTN13 / P grac -SP sacB -4GT, pHTN13 / P grac -SP lytF -4GT, pHTN13 / P grac -SP yybN -4GT, pHTN13 / P grac -SP ybbE -4GT, pHTN13 / P grac -SP ydbK -4GT, pHTN13 / P grac -SP yddT -4GT, pHTN13 / P grac -SP phrC -4GT, pHTN13 / P grac -SP cccA -4GT, pHTN13 / P grac -SP yxiT -4GT, pHTN13 / P grac -SP yvbX -4GT, pHTN13 / P 43 -SP samyQ -4GT, pHTN13 / P 556 -SP samyQ -4GT, pHTN13 / P spovG -SP samyQ -4GT, pHTN13 / P yvyD -SP samyQ -4GT, pHTN13 / P haG -SP samyQ -4GT, pHTN13 / P lytR -SP samyQ -4GT, pHTN13 / P sigW -SP samyQ -4GT, pHTN13 / P xylA -SP samyQ -4GT, pHTN13 / P yqfD -SP samyQ -4GT, pHTN13 / P mmgA -SP samyQ -4GT, pHTN13 / P gerBC -SP samyQ -4GT, pHTN13 / P bsamy -SP samyQ -4GT, pHTN13 / P phrC -SP samyQ -4GT, pHTN13 / P gsiB -SP samyQ -4GT, pHTN13 / P nprE -SP samyQ -4GT, pHTN13 / P bltD -SP samyQ -4GT, pHTN13 / P spovG -P haG -SP samyQ -4GT, pHTN13 / P spovG -P yvyD -SP samyQ -4GT, pHTN13 / P spovG -P 43 -SP samyQ -4GT, pHTN13 / P spovG -P spovG -SP samyQ -4GT, pHTN13 / P spovG -P spovG -SP sacB One or more of -4GT.

9. The recombinant Bacillus subtilis engineered strain as described in any one of claims 1-3, or the method as described in any one of claims 4-6, or the culture medium as described in claim 7, or the substance as described in claim 8, in the fields of fermentation production of 4-α-glycosyltransferase, preparation of high-yield 4-α-glycosyltransferase 4GT Bacillus subtilis genetically engineered strains, high-efficiency fermentation production, large-scale / economical production of 4GT enzyme preparations, efficient secretion expression system, starch modification and functional food development, and food and pharmaceutical fields.

10. The recombinant Bacillus subtilis engineered strain, method, culture medium, or substance according to any one of claims 1-9, characterized in that, The gene encoding the 4-α-glycosyltransferase is derived from *Thermusthermophilus* HB8, ATCC27634; and / or, The nucleotide sequence of the gene encoding the 4-α-glycosyltransferase is shown in SEQ ID NO: 1; the SP samyQ The signal peptide has an amino acid sequence identical to positions 1 to 31 of the α-amylase precursor protein shown in NCBI accession number AAA22192.1; the SP sacB The signal peptide has an amino acid sequence identical to positions 1 to 29 of the SacB protein shown in NCBI accession number CCD10844.1; the SP yybN The signal peptide has an amino acid sequence identical to positions 1 to 30 of the DUF2712 domain protein shown in NCBI accession number WP_009968442.1; the SP cccA The signal peptide has an amino acid sequence identical to positions 1 to 27 of the cytochrome c-550 protein shown in NCBI accession number MCY9210509.1; the SP lytF The signal peptide has an amino acid sequence identical to that shown in NCBI accession number AFG28268.1; the SP ybbE The signal peptide has an amino acid sequence identical to that shown in NCBI accession number AFG28210.1; the SP ydbK The signal peptide has an amino acid sequence identical to that shown in NCBI accession number AFG28261.1; the SP yddT The signal peptide has an amino acid sequence identical to that shown in NCBI accession number AFG28204.1; the SP phrC The signal peptide has an amino acid sequence identical to that shown in NCBI accession number AFG28211.1; the SP yxiT The signal peptide has an amino acid sequence identical to that shown in NCBI accession number AFG28238.1; the SP yvbX The signal peptide has the same amino acid sequence as that shown in NCBI accession number AFG28232.1; The P grac The promoter has a nucleotide sequence identical to that of positions 613110 to 613244 in the sequence shown in NCBI accession number CP133412.1; the P 43 The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP035167.1, from position 2449187 to 2449486; the P 556 The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP133412.1, from positions 3343655 to 3343709; the P spovG The promoter has a nucleotide sequence identical to positions 55546 to 55845 of the sequence shown in NCBI accession number CP133412.1; the P yvyD The promoter has a nucleotide sequence identical to positions 3926909 to 3927208 of the sequence shown in NCBI accession number CP154918.1; the P haG The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP133412.1, from position 3433573 to 3433872; the P lytR The promoter has a nucleotide sequence identical to positions 2507534 to 2507833 of the sequence shown in NCBI accession number CP116829.1; the P sigW The promoter has a nucleotide sequence identical to that of positions 188854 to 189153 in the sequence shown in NCBI accession number CP035411.1; the P xylA The promoter has a nucleotide sequence identical to positions 1734205 to 1734714 of the sequence shown in NCBI accession number CP133412.1; the P yqfD The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP035411.1, from positions 2481808 to 2482107; the P mmgA The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP133412.1, from position 2356834 to 2357133; the P gerBC The promoter has a nucleotide sequence identical to positions 3486142 to 3486441 of the sequence shown in NCBI accession number CP133412.1; the P bsamy The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP054050.1, from position 3852127 to 3852326; the P phrC The promoter has a nucleotide sequence identical to positions 413520 to 413819 of the sequence shown in NCBI accession number CP133412.1; the P gsiB The promoter has a nucleotide sequence identical to that of positions 478116 to 478421 in the sequence shown in NCBI accession number CP133412.1; the P nprE The promoter has a nucleotide sequence identical to the complementary strand of the sequence shown in NCBI accession number CP035411.1 from position 1518759 to 1519160; the P bltD The promoter has the same nucleotide sequence as positions 2514262 to 2514562 in the sequence shown in NCBI accession number CP133412.1.

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